Polymer nanoparticle, polymer composition, method of making a polymer nanoparticle, method for treatment of bacterial biofilms, and method for detection of bacterial biofilms

ABSTRACT

A polymer nanoparticle includes a polymer having repeating units of formula (I) wherein X, L 1 , and R 1  are as defined herein. Methods of preparing the polymer nanoparticles and compositions comprising the nanoparticles are also disclosed. The polymers nanoparticles described herein are particularly useful for the treatment of bacterial biofilms.

BACKGROUND

Bacterial biofilms are highly resilient microbial assemblies that aredifficult to eradicate. See, e.g., Costerton, J. W.; Stewart, P. S.;Greenburg, E. P. Bacterial Biofilms: A Common Cause of PersistentInfections. Science 1999, 284, 1318-1322. These robust biofilmsfrequently occur on synthetic implants and indwelling medical devicesincluding urinary catheters, arthro-prostheses, and dental implants.See, e.g., Lindsay, D.; von Holy, A. Bacterial Biofilms within theClinical Setting: What Healthcare Professionals Should Know. J. Hosp.Infect. 2006, 64, 313-325; Costerto Bacterial biofilms are highlyresilient microbial assemblies that are difficult to eradicate. See,e.g., Costerton, J. W.; Stewart, P. S.; Greenburg, E. P. BacterialBiofilms: A Common Cause of Persistent Infections. Science 1999, 284,1318-1322. These robust biofilms frequently occur on synthetic implantsand indwelling medical devices including urinary catheters,arthro-prostheses, and dental implants. See, e.g., Lindsay, D.; vonHoly, A. Bacterial Biofilms within the Clinical Setting: What HealthcareProfessionals Should Know. J. Hosp. Infect. 2006, 64, 313-325;Costerton, J. W.; Montanaro, L.; Arciola, C. R. Biofilm in ImplantInfections: Its Production and Regulation. Int. J. Artif. Organs 2005,28, 1062-1068; Busscher, H. J.; Rinastiti, M.; Siswomihardjo, W.; vander Mei, H. C. Biofilm Formation on Dental Restorative and ImplantMaterials. J. Dent. Res. 2010, 89, 657-665. Biofilm proliferation canalso occur on dead or living tissues, leading to endocarditis, otitismedia, and chronic wounds. See, e.g., Costerton, W.; Veeh, R.;Shirtliff, M.; Pasmore, M.; Post, C.; Ehrlich, G. The Application ofBiofilm Science to the Study and Control of Chronic BacterialInfections. J. Clin. Invest. 2003, 112, 1466-1477; Ehrlich, G.; Veeh,R.; Wang, X.; Costerton, J. W.; Hayes, J. D.; Hu, F. Z.; Daigle, B. J.;Ehrlich, M. D.; Post, J. C. Mucosal Biofilm Formation on Middle-EarMucosa in the Chinchilla Model of Otitis Media. JAMA 2002, 287, 1710;James, G. A; Swogger, E.; Wolcott, R.; Pulcini, E. deLancey; Secor, P.;Sestrich, J.; Costerton, J. W.; Stewart, P. S. Biofilms in ChronicWounds. Wound Repair Regen. 2007, 16, 37-44. The persistent infectionsand their concomitant diseases are challenging to treat, as biofilmsdevelop a high resistance to host immune responses and the extracellularpolymeric substances limit antibiotic penetration into biofilms. See,e.g., Stewart, P. S.; Costerton, J. W. Antibiotic Resistance of Bacteriain Biofilms. Lancet 2001, 358, 135-138; Szomolay, B.; Klapper, I.;Dockery, J.; Stewart, P. S. Adaptive Responses to Antimicrobial Agentsin Biofilms. Environ. Microbiol. 2005, 7, 1186-1191. Current techniquesto remove biofilms on man-made surfaces include disinfecting the surfacewith bleach or other caustic agents. See, e.g., Marion-Ferey, K.;Pasmore, M.; Stoodley, P.; Wilson, S.; Husson, G. P.; Costerton, J. W.Biofilm Removal from Silicone Tubing: An Assessment of the Efficacy ofDialysis Machine Decontamination Procedures Using an in Vitro Model. J.Hosp. Infect. 2003, 53, 64-71. Biofilms in biomedical contexts are verychallenging, with therapies based on excising infected tissues combinedwith long-term antibiotic therapy, incurring high health care costs andlow patient compliance due to the invasive treatment. See, e.g., Lynch,A. S.; Robertson, G. T. Bacterial and Fungal Biofilm Infections. Annu.Rev. Med. 2008, 59, 415-428. This issue is exacerbated by theexponential rise in antibiotic resistant bacteria. See, e.g., Levy, S.B.; Marshall, B. Antibacterial Resistance Worldwide: Causes, Challengesand Responses. Nat. Med. 2004, 10, S122-S129.

While synthetic materials such as nanoparticles and polymers whichexhibit broad spectrum activity against bacterial species exist, lack ofspecificity and toxicity towards mammalian cells limit their use inbiological settings. Accordingly, there remains a continuing need in theart to develop new synthetic platforms which can effectively treatbiofilms within a human host without causing adverse side effects suchas the hemolysis of red blood cells.

BRIEF SUMMARY

One embodiment is a polymer nanoparticle comprising a polymer comprisingrepeating units of formula (I)

wherein X is independently at each occurrence —O—, —S—, —CH₂—,—(CR⁴R⁵)—, or

wherein R⁴ and R⁵ are independently at each occurrence a C₁₋₆ alkylgroup and R⁶ and R⁷ are independently at each occurrence hydrogen or aC₁₋₆ alkyl group; L¹ is independently at each occurrence a divalentgroup that is (—CH₂—)_(z), wherein z is an integer from 3 to 18; and R¹is independently at each occurrence an ammonium group, a phosphoniumgroup, a zwitterionic group, a carboxylate group, a sulfonate group, analkylene oxide group, or a combination thereof.

Another embodiment is a method of making the polymer nanoparticle, themethod comprising combining the polymer comprising repeating units offormula (I) and an aqueous solution.

Another embodiment is a polymer comprising repeating units of formula(I)

wherein X is independently at each occurrence —O—, —S—, —CH₂—,—(CR⁴R⁵)—, or

wherein R⁴ and R⁵ are independently at each occurrence a C₁₋₆ alkylgroup and R⁶ and R⁷ are independently at each occurrence hydrogen or aC₁₋₆ alkyl group; L¹ is independently at each occurrence a divalentgroup that is (—CH₂—)_(z), wherein z is an integer from 3 to 18; and R¹is independently at each occurrence an ammonium group, a phosphoniumgroup, a zwitterionic group, a carboxylate group, a sulfonate group, analkylene oxide group, or a combination thereof.

Another embodiment is a method of treating a bacterial biofilm, themethod comprising contacting an aqueous composition comprising aplurality of polymer nanoparticles or the polymer with a bacterialbiofilm.

Another embodiment is a method for detecting a bacterial biofilm, themethod comprising contacting an aqueous composition comprising aplurality of polymer nanoparticles comprising a copolymer comprisingrepeating units of formula (I) and (IX)

wherein X is independently at each occurrence —O—, —S—, —CH₂—,—(CR⁴R⁵)—, or

wherein R⁴ and R⁵ are independently at each occurrence a C₁₋₆ alkylgroup and R⁶ and R⁷ are independently at each occurrence hydrogen or aC₁₋₆ alkyl group; L¹ is independently at each occurrence a divalentgroup that is (—CH₂—)_(z), wherein z is an integer from 3 to 18; R¹ isindependently at each occurrence an ammonium group, a phosphonium group,a zwitterionic group, a carboxylate group, a sulfonate group, analkylene oxide group, or a combination thereof; Z is a divalent C₆₋₂₀arylene group, a divalent C₁₋₂₀ alkylene oxide group, a divalentpoly(C₁₋₆ alkylene oxide) group, or an amino acid containing group; y is0 or 1; and R⁸ is a fluorescent group; with a surface; and measuringfluorescence, wherein the presence of fluorescence is indicative of thepresence of a bacterial biofilm.

These and other embodiments are described in detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Figures are exemplary embodiments.

FIG. 1 is a schematic illustration of polymer self-assembly into polymernanoparticles.

FIG. 2 is a chemical scheme illustrating the synthesis of compounds 1,2, and 3.

FIG. 3 is a chemical scheme illustrating the synthesis of compounds 4and 5.

FIG. 4 is a chemical scheme illustrating the synthesis of polymers 6 and7.

FIG. 5 is a chemical scheme illustrating the synthesis of polymers 8 and9.

FIG. 6 is a transmission electron micrograph (TEM) of polymernanoparticles. The scale bar is 100 nanometers (nm).

FIG. 7 shows a particle size distribution of the polymer nanoparticlesobtained by dynamic light scattering (DLS).

FIG. 8 is a schematic illustration of the Förster Resonance EnergyTransfer (FRET) results obtained for the polymer nanoparticles, as wellas a schematic illustration of the FRET process.

FIG. 9 shows the viability of a P. aeruginosa ATC-19660 biofilm whencontacted with varying concentrations of polymer nanoparticles.

FIG. 10 shows the viability of a S. aureus (MRSA) biofilm with varyingconcentrations of polymer nanoparticles.

FIG. 11 shows the viability of a P. aeruginosa CD-1006 biofilm withvarying concentrations of polymer nanoparticles.

FIG. 12 shows the viability of a E. cloacae CD-1412 biofilm with varyingconcentrations of polymer nanoparticles.

FIG. 13 shows the viability of macrophages with varying concentrationsof polymer nanoparticles.

FIG. 14 shows relative TNF-α expression with varying concentrations ofpolymer nanoparticles.

FIG. 15 shows toxicity of polymer nanoparticles to P. aeruginosa(ATCC-19660) biofilms that are grown in the presence of 3T3 fibroblastcells.

FIG. 16 shows resistance development during serial passaging in thepresence of sub-MIC levels of antimicrobials. The y-axis is the highestconcentration the cells grew in during passaging. FIG. 16 isrepresentative of three independent experiments.

FIG. 17 illustrates the mechanism of fluorescence for pH-sensitivefluorophore tagged polymers, showing that the quenched dye will recoverits fluorescence in acidic environments.

FIG. 18 shows confocal microscopy images indicating fluorescencerecovery of the fluorescent dye as the polymer nanoparticles penetratethe biofilm. The negative control of polymer without dye (middle row)shows no green fluorescence. Scale bars are 30 micrometers.

FIG. 19 is a plot of normalized fluorescence intensities with respect tobiofilm depth.

FIG. 20 is a plot showing the region in which therapeutic synergy can beobtained for the polymer nanoparticles and the antibiotic colistin.

FIG. 21 shows confocal microscopy images showing that when polymernanoparticles are incubated with colistin, enhanced colistin uptake isobserved. Scale bars are 30 micrometers.

FIG. 22 shows quantitative analysis of colistin uptake with and withoutpolymer based on normalized rhodamine-G-labelled colistin fluorescence.

DETAILED DESCRIPTION

The present inventors have developed a synthetic route to spontaneouslygenerate norbornene-based polymeric nanoparticles in aqueousenvironments. The strategy described herein involves incorporatinghomopolymers having long alkyl chains between the norbornene backbonesand the terminal headgroups. It is believed that the self-assembly ofthese polymers into nanoparticles is dictated via the hydrophobiceffect, similar to traditional diblock polymeric micelles. Withoutwishing to be bound by theory, it is believed that the polymers canassemble and bury the long alkyl chains while exposing the chargedterminal head group of each repeat unit. The polymer nanoparticlesdescribed herein can address the lack of specificity and toxicitytowards mammalian cells typical of other known synthetic systems. Thepolymeric nanoparticles described herein possess low minimal inhibitorconcentrations (MICs) in the nanomolar range towards pathogenicplanktonic bacteria while maintaining remarkable hemolytic activities.Additionally, the present inventors have demonstrated the effectivekilling of pathogenic biofilms, not yet observed with otherpolymer-based systems. The polymeric nanoparticles can eradicatebiofilms, while maintaining a high therapeutic index against red bloodcells. Advantageously, the polymeric nanoparticles described herein donot require the presence of organic solvents during preparation, andadditional purification is also not required, saving both time andmoney. In addition, consistent nanoparticle size is possible compared totraditional micelle formation strategies. In another advantageousfeature, the present inventors have shown that further conjugating afluorescent dye molecule to the polymer backbone used to form thepolymer nanoparticles can be particularly useful for imaging thebiofilms, with potential applications for in vivo imaging. Furthermore,the present inventors have also discovered co-incubation of the polymernanoparticles and an antibiotic can provide a therapeutic synergy,enhancing antibiotic uptake into the biofilms for improved therapeuticefficacy.

Accordingly, one aspect of the present disclosure is a polymernanoparticle. The polymer nanoparticles can have a diameter of 1 to 100nanometers, or 1 to 50 nanometers, or 1 to 25, nanometers, or 10 to 20nanometers. The diameter of the particles can be measured using knowntechniques, for example, by measuring the diameter observed bytransmission electron microscopy (TEM). There is no particularrestriction on the shape of the polymer nanoparticles, which can bedictated, for example, by polymer composition, polymer molecular weight,polymer concentration, and the like. In some embodiments, the polymernanoparticles can be substantially spherical.

The polymer nanoparticles comprise a polymer comprising repeating unitsof formula (I)

wherein X is independently at each occurrence —O—, —S—, —CH₂—,—(CR⁴R⁵)—, or

wherein R⁴ and R⁵ are independently at each occurrence a C₁₋₆ alkylgroup and R⁶ and R⁷ are independently at each occurrence hydrogen or aC₁₋₆ alkyl group; L¹ is independently at each occurrence a divalentgroup that is (—CH₂—)_(z), wherein z is an integer from 3 to 18 (e.g.,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18); and R¹ isindependently at each occurrence an ammonium group, a phosphonium group,a zwitterionic group, a carboxylate group, a sulfonate group, analkylene oxide group, or a combination thereof. In some embodiments, thepolymer nanoparticle comprises a polymer that is a homopolymerconsisting of repeating units of formula (I).

In some embodiments, X in formula (I) can be —O—. In some embodiments, Xin formula (I) can be —CH₂—. In some embodiments, L¹ in formula (I) is adivalent group that is (—CH₂—)_(z), wherein z is an integer from 6 to 12(e.g., 6, 7, 8, 9, 10, 11, or 12).

As described above, R¹ can be an ammonium group, a phosphonium group, azwitterionic group, a carboxylate group, a sulfonate group, an alkyleneoxide group, or a combination thereof. In some embodiments, R¹ is anammonium group. For example, in some embodiments, R¹ is an ammoniumgroup of formula (II)

wherein R² is a C₁₋₁₂ alkyl group or a C₇₋₂₀ alkylaryl group, and Y isbromide, chloride, fluoride, iodide, hydroxide, phosphate, sulfonate,carbonate, acetate, hexafluorophosphate, tetrafluoroborate, mesylate,trifluoroacetate, p-toluenesulfonate, or a combination thereof. In someembodiments, Y is bromide, hydroxide, or a combination thereof. In someembodiments, R² is preferably a C₁₋₁₂ alkyl group, more preferably aC₁₋₆ alkyl group, or a benzyl group. For example, the ammonium group canbe selected from the following ammonium groups:

wherein Y is as defined above.

In some embodiments, R¹ can be a phosphonium group, for example, aphosphonium group according to formula (III)

wherein R³, R⁴, and R⁵ are independently at each occurrence a C₁₋₁₂alkyl group, a C₃₋₁₂ cycloalkyl group, or a C₆₋₂₀ aryl group, and Y isbromide, chloride, fluoride, iodide, hydroxide, phosphate, sulfonate,carbonate, acetate, hexafluorophosphate, tetrafluoroborate, mesylate,trifluoroacetate, p-toluenesulfonate, or a combination thereof. In someembodiments, Y is bromide, hydroxide, or a combination thereof. R³, R⁴,and R⁵ can be the same or different. In some embodiments, each of R³,R⁴, and R⁵ are a C₁₋₁₂ alkyl group, preferably a C₁₋₆ alkyl group,including linear and branched alkyl groups. In some embodiments, each ofR³, R⁴, and R⁵ are a C₆₋₁₂ aryl group. For example, the phosphoniumgroup can be selected from the following phosphonium groups:

wherein Y is as defined above.

In some embodiments, R¹ can be a zwitterionic group. A zwitterionicgroup is a group of the formula -A-B-C, wherein A is a center ofpermanent positive charge or a center of permanent negative charge, B isa divalent group comprising a C₁₋₁₂ alkylene group, a C₆₋₃₀ arylenegroup, or an alkylene oxide group, and C is a center of permanentnegative charge or a center of permanent positive charge, provided thatthe zwitterion has an overall net charge of zero (i.e., the zwitterionis net neutral). For example, in an embodiment wherein A is a center ofpermanent positive charge, C is a center of permanent negative charge.For example, in an embodiment wherein A is a center of permanentnegative charge, C is a center of permanent positive charge. In someembodiments, a center of permanent positive charge can include aquaternary ammonium group, a phosphonium group, a sulfonium group, andthe like. In some embodiments, the center of permanent positive chargeis preferably an ammonium group. In some embodiments, a center ofpermanent negative charge can include a sulfonate group, a phosphonategroup, a carboxylate group, a thiolate group, and the like. In someembodiments, the zwitterionic group is a sulfobetaine group, aphosphorylcholine group, or a carboxy betaine group. In an embodiment,the zwitterionic group is a sulfobetaine group wherein A is ammonium(e.g., a divalent dimethyl ammonium group (—N⁺(CH₃)₂—)), B is propyleneor butylene, and C is a sulfonate group (—SO₂O⁻). In an embodiment, thezwitterionic group is a carboxy betaine group wherein A is ammonium(e.g., a divalent dimethyl ammonium group (—N⁺(CH₃)₂—)), B is methyleneor ethylene, and C is a carboxylate group (—COO⁻). In an embodiments,the zwitterionic group is a phosphorylcholine group wherein A is aphosphonate, B is ethylene, and C is an ammonium group. For example, thezwitterionic group can be selected from the following zwitterionicgroups:

In some embodiments, R¹ can be a carboxylate group, for example acarboxylate group of formula (IV)

wherein L² is a group comprising a C₁₋₁₂ alkylene group, a C₆₋₃₀ arylenegroup, or an alkylene oxide group, preferably a C₁₋₁₂ alkylene group,more preferably a C₁₋₆ alkylene group. Y¹ is a cationic group, forexample sodium, potassium, calcium, magnesium, ammonium (NH₄+), aquaternary ammonium (e.g., N(CH₃)₄+), triethylammonium,diisopropylethylammonium, or a combination thereof.

In some embodiments, R¹ can be a sulfonate group, for example, asulfonate group of formula (V)

wherein L² and Y¹ can be as described above for formula (IV).

In some embodiments, R¹ can be a group according to formula (VI)

wherein L² and Y¹ can be as described above for formula (IV).

In some embodiments, R¹ can be an alkylene oxide group or apoly(alkylene oxide) group. For example, R¹ can be a group according toformula (VII)

wherein R⁶ is a C₁₋₆ alkyl group, preferably a methyl group, or ahydrogen, and n is an integer from 4 to 100.

In some embodiments, R¹ is preferably an ammonium group according toformula (II).

In some embodiments, R¹ is not a guanidinium group. In some embodiments,R¹ is not a pyridinium group. In some embodiments, the polymer excludesrepeating units which include one or more guanidinium groups orpyridinium groups.

In some embodiments, the polymer can optionally further compriserepeating units of formula (VIII)

wherein X is independently at each occurrence —O—, —S—, —CH₂—,—(CR⁴R⁵)—, or

wherein R⁴ and R⁵ are independently at each occurrence a C₁₋₆ alkylgroup and R⁶ and R⁷ are independently at each occurrence hydrogen or aC₁₋₆ alkyl group; and L³ is independently at each occurrence a C₃₋₂₀alkyl group. In some embodiments, X in formula (I) can be —O—. In someembodiments, X in formula (I) can be —CH₂—. L³ can be a branched orstraight chain alkyl group, and can be substituted or unsubstituted.Exemplary L³ alkyl groups can include, for example, n-propyl, n-butyl,n-hexyl, 2-ethylhexyl, 2-hexyldecyl, 2-octyldodecyl, and the like. Insome embodiments, L³ is preferably a branched alkyl group. In someembodiments, L³ is preferably 2-ethylhexyl, 2-hexyldecyl,2-octyldodecyl, and the like, more preferably 2-ethylhexyl.

When present, the polymer can include repeating units of formula (VIII)in an amount of greater than 0 to 25 mole percent, based on the totalmoles of repeating units in the polymer. For example, the polymer caninclude repeating units of formula (VIII) in an amount of 1 to 25 molepercent, or 5 to 25 mole percent, or 10 to 25 mole percent, or 10 to 20mole percent. In some embodiments, the polymer does not include anyrepeating units according to formula (VIII).

In some embodiments, the polymer further comprises repeating units offormula (IX)

wherein X is independently at each occurrence —O—, —S—, —CH₂—,—(CR⁴R⁵)—, or

wherein R⁴ and R⁵ are independently at each occurrence a C₁₋₆ alkylgroup and R⁶ and R⁷ are independently at each occurrence hydrogen or aC₁₋₆ alkyl group; L¹ is independently at each occurrence a divalentgroup that is (—CH₂—)_(z), wherein z is an integer from 3 to 18; Z is adivalent C₆₋₂₀ arylene group, a divalent C₁₋₂₀ alkylene oxide group, adivalent poly(C₁₋₆ alkylene oxide) group, or an amino acid containinggroup; y is 0 or 1; and R⁸ is a fluorescent group. Z and R⁸ arecovalently bonded through a pH sensitive imine linkage. Thus, the groupZ is derived from a moiety including a primary amine capable of formingthe pH sensitive imine bond to fluorescent dye R⁸ which includes analdehyde group. In some embodiments, y is 0, and linking group L¹ isdirectly connected to the fluorescent group R⁸ via the imine linkage. Inother embodiments, y is 1. In some embodiments, Z can be a C₆₋₂₄monocyclic or polycyclic aromatic group that is substituted with atleast one primary amine group. The aromatic group Z can be bonded tolinking group L¹, for example, through an ether bond. For example, thearomatic group Z can be

In some embodiments, Z is preferably

In some embodiments, Z can be an amino acid-containing group, forexample, having the structure

In some embodiments, Z can include a divalent C₁₋₂₀ alkylene oxide groupor a divalent poly(C₁₋₆ alkylene oxide) group, wherein Z is linked to L¹via an ether linkage. Exemplary Z groups of this type can include

wherein p is an integer from 3 to 10.

The fluorescent dye R⁸ can generally be any fluorescent dye thatincludes the functional group as described above needed to form the pHsensitive linkage with group Z, provided that the excitation andemission wavelengths of the dye fall within the region of interest, ascan be determined by a person skilled in the art depending on thedesired application. In some embodiments, the dye can have an emissionin the visible of near-infrared (NIR) spectrum). Exemplary dyes that canbe useful include, but are not limited to, carbocyanine,indocarbocyanine, oxacarbocyanine, thuicarbocyanine and merocyanine,polymethine, coumarine, rhodamine, xanthene, fluorescein,borondipyrromethane (BODIPY), Cy5, Cy5.5, Cy7, VivoTag-680,VivoTag-S680, VivoTag-S750, AlexaFluor660, AlexaFluor680, AlexaFluor700,AlexaFluor750, AlexaFluor790, Dy677, Dy676, Dy682, Dy752, Dy780,DyLight547, Dylight647, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor750, IRDye 800CW, IRDye 800RS, IRDye 700DX, ADS780WS, ADS830WS, andADS832WS. It is noted that the aforementioned dyes can be chemicallymodified to include an aldehyde group to facilitate conjugation to thepolymer, if needed. Such chemical modifications will be apparent to aperson skilled in the art. In some embodiments, the dye R⁸ is preferablya BODIPY dye. BODIPY (borondipyrromethane) dyes have a general structureof 4,4′-difluoro-4-bora-3a,4a-diaza-s-indacene) and sharp fluorescencewith high quantum yield and excellent thermal and photochemicalstability.

In a very specific embodiment, the polymer further comprises repeatingunits of formula (IXA)

In the above formula IXA, L¹ is a C₆ methylene group, Z is a linkerhaving the structure

andR⁸ is a fluorescent group having the structure

When present, the repeating units of formula (IX) are present in a molarratio of repeating units of formula (I):repeating units of formula (IX)of 15:1 to 5:1, or 12:1 to 7:1, or 11:1 to 8:1.

In some embodiments, the polymer consists of repeating units accordingto formula (I), and optionally, formula (VIII) or formula (IX).

In an embodiment, the polymer comprises repeating units according toformula (I), wherein X is —O—, L¹ is a divalent group that is(—CH₂—)_(z), wherein z is an integer from 6 to 12, and R¹ is an ammoniumgroup of formula (II), wherein R² is a C₁₋₆ alkyl group or a benzylgroup and Y is bromide, hydroxide, or a combination thereof.

In another embodiment, the polymer comprises repeating units accordingto formula (I), wherein X is —CH₂—, L¹ is a divalent group that is(—CH₂—)_(z), wherein z is an integer from 6 to 12, and R¹ is an ammoniumgroup of formula (II), wherein IV is a C₁₋₁₀ alkyl group and Y isbromide, hydroxide, or a combination thereof.

The polymer can have a number average molecular weight of 5,000 to100,000 grams per mole, or 5,000 to 50,000 grams per mole, or 10,000 to30,000 grams per mole. Number average molecular weight can be determinedusing gel permeation chromatography (GPC).

The polymer can generally be prepared using known techniques. Forexample, the polymer can be prepared by ring opening metathesispolymerization (ROMP) of a suitable cyclic olefin monomer (e.g., anappropriately functionalized norbornene, oxanorbornene, and derivativesthereof) in the presence of a ROMP catalyst such as aruthenium-containing catalyst. An example of such a procedure is furtherdescribed in the working examples below. Metal-free ROMP techniques canalso be used, for example, as describe in Ogawa, K. A.; Goetz, A. E.;Boydston, A. J. Metal-Free Ring-Opening Metathesis Polymerization. J.Am. Chem. Soc. 2015, 137, 1400-1403.

The polymer nanoparticles of the present disclosure can be formed by amethod comprising combining the above-described polymer having repeatingunits according to formula (I) and an aqueous solution. The aqueoussolution can comprise water, deionized water, a buffer (e.g., phosphatebuffered saline, phosphate buffer, and the like), and the like, or acombination thereof. In some embodiments, the polymer is added to theaqueous solution in an amount of 0.00001 to 10 weight percent, or0.00001 to 1 weight percent, or 0.0001 to 0.5 weight percent, or 0.0001to 0.1 weight percent, or 0.0001 to 0.01 weight percent, or 0.0001 to0.005 weight percent, or 0.0005 to 0.005 weight percent, based on theweight of the aqueous solution to provide the polymer nanoparticles. Insome embodiments, the polymer nanoparticles can be provided as acomposition in the aqueous solution. In some embodiments, the polymernanoparticles can be isolated from the aqueous solution following theirformation. Isolating the nanoparticles can be by, for example, dialysis,lyophilization, or a combination thereof. Advantageously, the polymerparticles can be isolated and stored as a powder, which can bereconstituted in an aqueous solution to provide an aqueous compositioncomprising the nanoparticle, when desired.

The aqueous composition comprising the nanoparticles can include 0.00001to 10 weight percent, or 0.00001 to 1 weight percent, or 0.0001 to 0.5weight percent, or 0.0001 to 0.1 weight percent, or 0.0001 to 0.01weight percent, or 0.0001 to 0.005 weight percent, or 0.0005 to 0.005weight percent of the polymer nanoparticles, based on the total weightof the aqueous composition. Accordingly, the aqueous composition caninclude 90 to 99.99999, such that the amount of the aqueous solution andthe polymer nanoparticles totals 100 weight percent.

In some embodiments, the aqueous composition comprising the polymernanoparticles can optionally further include one or more additives thatare generally known in the art, with the proviso that the additives donot significantly adversely affect one or more desired properties of thecomposition. Furthermore, it can be particularly desirable that thepresence of an additive does not significantly interfere with thestructure of the nanoparticles. Additives can include stabilizers,thickeners, viscosity enhancers, coloring agent, surfactants,emulsifiers, humectants, antibiotics, siderophores, quorum sensinginhibitors, and the like, or a combination thereof. In some embodiments,no additives are present in the aqueous composition.

In some embodiments, the polymer nanoparticles can optionally furtherinclude additives such as hydrophobic antibiotics. The presence of thehydrophobic antibiotic can increase the antimicrobial activity of thenanoparticle. Suitable hydrophobic antibiotics can include nalidixicacid, cinoxacin, norfloxacin, ciprofloxacin, enoxacin, ofloxacin,levofloxacin, sparfloxacin, moxifloxacin, gemifloxacin, trovafloxacin,ampicillin, amoxicillin, carbenicillin, carfecillin, ticarcillin,azlocillin, mezlocillin, piperacillin, cefepime, tetracycline,gentamicin, tobramycin, streptomycin, neomycin, kanamycin, amikacin,cefoselis, and cefquinome. When present, the hydrophobic antibiotic canbe included in the nanoparticle in an amount of 0.001 to 10 weightpercent, based on the weight of the nanoparticle.

Another aspect of the present disclosure is a method for treating abacterial biofilm. The method comprises contacting the above describedaqueous composition comprising a plurality of the polymer nanoparticlesor an aqueous composition comprising the above-described polymer with abacterial biofilm.

A “biofilm” refers to a population of bacteria attached to an inert orliving surface. Thus, biofilms can form on a counter, a table, waterpipes, implants, catheters, cardiac pacemakers, prosthetic joints,cerebrospinal fluid shunts, endotracheal tubes, and the like. In someembodiments, the biofilm can be present on a living surface, for exampleskin or in a wound, and on teeth (e.g., dental plaque). Bacteria in abiofilm are enmeshed in an extracellular polymer matrix, generally apolysaccharide matrix, which holds the bacteria together in a mass, andfirmly attaches the bacterial mass to the underlying surface. Evidencehas shown that biofilms constitute a significant threat to human health.Wounds and skin lesions are especially susceptible to bacterialinfection.

In some embodiments, the bacterial biofilm can be a gram-negativebacterial biofilm or a gram-positive bacterial biofilm. In someembodiments, the bacterial biofilm comprises Escherichia coli (e.g., E.coli DH5a), Pseudomonas bacteria (e.g., Pseudomonas aeruginosa),Staphylococcal bacteria (e.g., Staphylococcal aureus),Enterobacteriaceae bacteria (e.g., E. cloacae complex), Streptococcusbacteria, Haemophilus influenzae, Leptospira interrogans, Legionellabacteria, Micrococcus bacteria (e.g., Micrococcus luteus), Bacillusbacteria (e.g., Bacillus subtilis, Bacillus cereus, Bacilluslicheniformic, Bacillus megaterium), Burkholderia bacteria (e.g.,Burkholderia cepacia), Amycolatopsis bacteria (e.g., Amycolatopsisazurea), Mycobacterium bacteria (e.g., Mycobacterium tuberculosis),Acinetobacter bacteria (e.g., Acinetobacter baumannii), Enterococcusbacteria (e.g., Enterococcus faecium), Klebsiella bacteria (e.g.,Klebsiella pneumonia), Acinetobacter bacteria, or a combination thereof.

Contacting an aqueous composition comprising the nanoparticles or thepolymer with a biofilm can effectively kill bacterial cells present inthe biofilm. Thus, compositions prepared from the above-describednanoparticles and polymers can be particularly useful as disinfectantsor antimicrobial compositions. The contacting can be under conditionseffective to treat the biofilm, for example for a time of 10 minutes to5 hours, or 1 hour to 3 hours, and at a temperature of 25 to 37° C. Asused herein, “treating a biofilm” can refer to killing at least 20%, orat least 40%, or at least 50%, or at least 60%, or at least 80%, or atleast 90% of the bacterial cells present in the biofilm. In someembodiments, contacting the composition with a biofilm can completelyremove the biofilm (i.e., the dispersion is toxic to greater than 90%,or 99% or 99.9% of the bacterial cells of the biofilm upon contactingthe composition with the biofilm).

In some embodiments, the method can further comprise contacting anantibiotic with the bacterial biofilm. Contacting the antibiotic withthe bacterial biofilm can occur prior to, simultaneously with, or aftercontacting the bacterial biofilm with the aqueous composition comprisingthe polymer nanoparticles. Preferably, contacting the antibiotic withthe bacterial biofilm can occur simultaneously with the contacting ofthe aqueous composition with the bacterial biofilm (i.e., the polymernanoparticles and the antibiotic can be co-incubated with the bacterialbiofilm). The antibiotic can be in addition to any antibiotic that canbe present in the polymer nanoparticle formulation as described above.The antibiotic can be a hydrophilic antibiotic, for example, anaminoglycoside, a beta-lactam, a glycopeptide, colistin, and the like,or a combination thereof. In a specific embodiment, the antibiotic canbe colistin. Without wishing to be bound by theory, the contacting of abacterial biofilm with both the polymer nanoparticles and an antibiotic(e.g., colistin) is believed to provide enhance treatment of thebacterial biofilm via a synergistic effect resulting from theco-delivery, as will be discussed further in the working examples below.

Another aspect of the present disclosure is a method for detecting abacterial biofilm. The method comprises contacting an aqueouscomposition comprising a plurality of polymer nanoparticles comprising acopolymer comprising repeating units of formula (I) and (IX), asdescribed above, with a surface. The aqueous composition can be asdescribed above in relation to the method for treating a bacterialbiofilm. The surface can be, for example, a counter, a table, waterpipes, implants, catheters, cardiac pacemakers, prosthetic joints,cerebrospinal fluid shunts, endotracheal tubes, and the like. In someembodiments, the surface can be a living surface, for example skin, awound, or teeth. The method further comprises measuring fluorescence ofthe treated surface, where the presence of fluorescence is indicative ofthe presence of a bacterial biofilm on the surface. In some embodiments,the method can further be a method for treating the detected bacterialbiofilm, as contacting the aqueous composition with an infected surfacecan effectively kill bacterial cells present in the biofilm.

In summary, the present disclosure provides new polymers and polymernanoparticles prepared therefrom. The polymer nanoparticles demonstratehighly effective therapeutic behavior, successfully eradicatingpathogenic biofilm strains of clinical isolates. Thus, the nanoparticlesdescribed herein have potential applications as general surfacedisinfectants as well as antiseptics for wound treatment. The facileself-assembly strategy used to prepare the nanoparticles provides apromising platform to create effective delivery vehicles to combatbacterial biofilms. Furthermore, the therapeutic efficacy of the polymernanoparticles against bacterial biofilms can be enhanced by co-deliverywith an antibiotic.

The polymers, polymer nanoparticles, and methods described herein arefurther illustrated by the following non-limiting examples.

EXAMPLES

Experimental details for the preparation of the monomers, polymers, andnanoparticles used for the following examples are provided below.

Monomer Synthesis

Compound 1, shown in FIG. 2, was synthesized according to the followingprocedure. In a pressure tube, furan (4.5 milliliters, 61.7 millimoles,1.5 eq.) and maleimide (4.0 grams, 41.1 millimoles, 1.0 eq.) were addedto 5 milliliters of diethyl ether. The tube was sealed and heated at100° C. overnight. The pressure tube was then cooled to room temperatureand the formed solid was removed by filtration, and washed with copiousamounts of diethyl ether to isolate 1 as a white solid. Compound 1 wasused without further purification. Proton nuclear magnetic resonancespectroscopy (¹H NMR) was used to confirm the structure of compound 1.¹H NMR was conducted at 400 MHz using deuterated methanol (MeOD) as thesolvent. The chemical shifts are reported as parts per million (ppm)using tetramethylsilane (TMS) as a reference: 11.14 (s, 1H), 6.52 (s,2H), 5.12 (s, 2H), 2.85 (s, 2H).

Compound 2, shown in FIG. 2, was synthesized according to the followingprocedure. To a 250 milliliter round bottom flask equipped with a stirbar was added 60 milliliters of dimethyl formamide (DMF). Compound 1(3.76 grams, 22.7 millimoles, 1.0 eq.) was then added with potassiumcarbonate (12.59 grams, 91.1 millimoles, 4.0 eq.). The reaction mixturewas heated at 50° C. for five minutes. Potassium iodide (0.68 grams, 4.5millimoles, 0.2 eq.) and 11-bromoundecanol (6.00 grams, 23.9 millimoles,1.05 eq.) were added and the mixture was stirred at 50° C. overnight.The reaction mixture was subsequently cooled to room temperature,diluted to 150 milliliters with ethyl acetate, and washed with water(7×50 milliliters) and brine (1×50 milliliters). The organic layer wasdried with sodium sulfate, filtered, and concentrated by rotaryevaporation to yield Compound 2. Compound 2 was purified by sonicationof the solid in hexanes, followed by filtration. NMR was conducted at400 MHz using deuterated chloroform (CDCl₃) as the solvent. NMR (400MHz, CDCl₃) 6.44 (s, 2H), 5.19 (s, 2H), 3.55, (t, 2H), 3.49 (t, 2H),2.79 (s, 2H), 1.9 (s, 1H), 1.39 (m, 4H), 1.2 (m, 14H).

Compound 3, shown in FIG. 2, was synthesized according to the followingprocedure. To a 250 milliliter round bottom flask equipped with a stirbar was added compound 2 (2.64 grams, 7.87 millimoles, 1.0 eq.).Dichloromethane (DCM) (100 milliliters) was then added, withtetrabromomethane (3.13 grams, 9.44 millimoles, 1.2 eq.). The reactionwas cooled to 0° C. using an ice bath. Finally, triphenylphosphine wasadded in portions (2.47 grams, 9.44 millimoles, 1.2 eq.) and allowed tostir for three hours. The reaction mixture was then concentrated byrotary evaporation and ethyl ether was added (200 milliliters) andplaced in the freezer for 2 hours to precipitate out triphenylphosphineoxide byproduct. The mixture was filtered and the filtrate wasconcentrated by rotary evaporation. Column chromatography was performedto yield compound 3, as a white solid. ¹H NMR (400 MHz, CDCl₃) 6.51 (s,2H), 5.27 (s, 2H), 3.45 (t, 2H), 3.41 (t, 2H), 2.83 (s, 2H), 1.85 (q,2H), 1.55 (q, 2H), 1.41 (q, 2H), 1.29 (m, 12H).

Compound 4, shown in FIG. 3, was synthesized according to the followingprocedure. To a 500 milliliter round bottom flask equipped with a stirbar was added cis-5-Norbornene-endo-2,3-dicarboxylic anhydride (12.0grams, 73.1 millimoles, 1.0 eq.). Next, toluene was added (250milliliters). Finally, 6-amino-1-hexanol (9.0 grams, 76.8 millimoles,1.05 eq.) was added and the reaction was stirred at reflux using adean-stark trap overnight. Afterwards, the reaction mixture wasconcentrated by rotary evaporation, and the residue was dissolved intoethyl acetate (100 milliliters). The ethyl acetate was extracted using 1Molar hydrochloric acid (HCl) (50 milliliters) followed by brine (50milliliters). The organic layer was and dried, filtered, andconcentrated by rotary evaporation to yield compound 4. No furtherpurification was performed. ¹H NMR (400 MHz, CDCl₃) 6.07 (s, 2H), 3.6(t, 2H), 3.35 (br, 2H), 3.29 (t, 2H), 3.21 (br, 2H), 1.7 (t, 2H), 1.51(m, 3H), 1.4 (m, 2H), 1.31 (m, 2H), 1.22 (m, 2H).

Compound 5, shown in FIG. 3, was synthesized according to the followingprocedure. To a 250 milliliter round bottom flask equipped with a stirbar was added 4 (5.0 grams, 19.0 millimoles, 1.0 eq.). Next, DCM (100milliliters) was added along with tetrabromomethane (7.87 grams, 23.75millimoles, 1.25 eq.). The reaction was cooled to 0° C. using an icebath. Finally, triphenylphosphine was added in portions (7.48 grams,28.5 millimoles, 1.50 eq.) and allowed to stir for three hours.Afterwards, the reaction mixture was concentrated by rotary evaporationand ethyl ether was added (200 milliliters) and placed in the freezerfor 2 hours to precipitate out triphenylphosphine oxide byproduct. Thereaction mixture was filtered and the filtrate was concentrated byrotary evaporation. Column chromatography was performed to yield 5, alight yellow oil. ¹H NMR (400 MHz, CDCl₃) 6.1 (s, 2H), 3.39 (t, 2H),3.38 (s, 2H), 3.31 (t, 2H), 3.25 (s, 2H), 1.83 (q, 2H), 1.72 (d, 1H),1.54 (d, 1H), 1.44 (m, 4H), 1.28 (m, 2H).

Polymer Synthesis

Polymer 6, shown in FIG. 4, was synthesized according to the followingprocedure. To a 10 milliliter pear-shaped air-free flask equipped with astir bar was added 3 (800 milligrams, 2.0 millimoles, 1.0 eq.) and 4milliliters of DCM. In a separate 10 milliliter pear-shaped air-freeflask was added Grubbs 3^(rd) generation catalyst (35.4 milligrams, 0.04millimoles, 0.02 eq.) and 1 milliliter of DCM. Both flasks were sealedwith septa and attached to a schlenk nitrogen/vaccum line. Both flaskswere freeze-pump-thawed three times each. After thawing, Grubbs 3^(rd)generation catalyst was syringed out and quickly added to the flaskcontaining 3 and allowed to react for 10 minutes. After the allottedtime, ethyl vinyl ether (200 microliters) was added and allowed to stirfor 15 minutes. Afterwards, the reaction was diluted to two times thevolume and precipitated into a heavily stirred solution of hexane (300milliliters). The precipitated polymer was filtered and dissolved intotetrahydrofuran (THF). The polymer was precipitated again into hexaneand filtered to yield 6. The polymer molecular weight was characterizedby gel permeation chromatography (GPC) against polystyrene standardseluting with tetrahydrofuran. Polymer 6 was found to have a weightaverage molecular weight (Mw) of 25,698 grams per mole, and apolydispersity (PDI) of 1.04. The polymer was also characterized using¹H NMR spectroscopy. ¹H NMR (400 MHz, CDCl₃) 6.0 (br, 1H), 5.7 (br, 1H),4.95 (br, 1H), 4.4 (br, 1H), 3.4 (br, 2H), 3.25 (br, 2H), 1.79 (q, 2H),1.5 (br, 2H), 1.34 (br, 2H), 1.2 (br, 14H).

Quaternary ammonium-containing polymer 7, shown in FIG. 4, wassynthesized according to the following procedure. Polymer 6 (50milligrams) was added to a 20 milliliter vial equipped with a stir bar.Next, excess of the necessary tertiary amines was added (10 millilitersof a 1 Molar trimethylamine solution in THF, all other amines were addedin an amount of 200 milligrams) to the vial and purged with nitrogen.First stage of the reactions involved stirring for 30 minutes at 80° C.The polymers precipitated during this time. Approximately half of theTHF was evaporated and replaced with methanol which re-dissolved thepolymers. The reaction was allowed to proceed overnight at 50° C.Afterwards, the solvent was completely evaporated, washed with hexane 2times, and dissolved into a minimal amount of water. The polymers wereadded to 10,000 molecular weight cut off (MWCO) dialysis membranes andallowed to stir for 3 days, changing the water periodically. The polymersolutions were filtered through PES syringe filters, and freeze-dried toyield the respective quaternary ammonium polymers 7. NMR indicatedcomplete conversion into the quaternary ammonium salts.

Polymer 8, shown in FIG. 5, was synthesized according to the followingprocedure. To a 10 milliliter pear-shaped air-free flask equipped with astirbar was added compound 5 (800 milligrams, 2.46 millimoles, 1.0 eq.)and 5 milliliters of DCM. In a separate 10 milliliter pear-shapedair-free flask was added Grubbs 2^(nd) generation catalyst (20milligrams, 0.047 millimoles, 0.02 eq.) and 1 milliliter of DCM. Bothflasks were sealed with septa and attached to a schlenk nitrogen/vacuumline. Both flasks were freeze-pump-thawed three times. After thawing,Grubbs 2^(nd) generation catalyst was syringed out and quickly added tothe flask containing 5 and allowed to react for 10 minutes. After theallotted time, ethyl vinyl ether (200 microliters) was added and allowedto stir for 15 minutes. The reaction was then diluted to two times thevolume and precipitated into a heavily stirred solution of hexane (300milliliters). The precipitated polymer was filtered and dissolved intoTHF. The polymer was precipitated again into hexane and filtered toyield polymer 8. Polymer 8 was found to have a Mw of 17,027 grams permole and a polydispersity of 1.36, as determined by GPC, eluting withTHF, and using a polystyrene calibration curve. ¹H NMR (400 MHz, CDCl₃)5.7 (br, 1H), 5.6 (br, 1H), 3.48 (br, 2H), 3.39 (br, 2H), 3.2 (br, 2H),2.95 (br, 2H), 1.85 (br, 2H), 1.55 (br, 2H), 1.45 (br, 6H).

Quaternary ammonium-containing polymer 9, shown in FIG. 5, wassynthesized according to the following procedure. Polymer 8 (50 mg) wasadded to 20 ml vials equipped with a stir bar. Next, excess of thenecessary tertiary amines was added (10 ml of a 1M trimethylaminesolution in THF, all other amines were 200 mg) to the vial and purgedwith nitrogen. First stage of the reactions involved stirring for 30minutes at 80° C. The polymers precipitated during this time. Half ofthe THF was evaporated and replaced with methanol which re-dissolved thepolymers. The reaction was allowed to proceed overnight at 50° C.Afterwards, the solvent was completely evaporated, washed with hexane 2times, and dissolved into a minimal amount of water. The polymers wereadded to 10,000 MWCO dialysis membranes and allowed to stir for 3 days,changing the water periodically. The polymers were filtered through PESsyringe filters, and freeze-dried to yield the respective quaternaryammonium polymers 9. NMR indicated complete conversion into thequaternary ammonium salts.

Polymer Nanoparticle Formation

Polymer nanoparticles were prepared from polymers 7 and 9. In general,the polymer (290 milligrams) was added to water (1 milliliter) tospontaneously form the polymer nanoparticles. The polymer nanoparticleswere imaged using transmission election microscopy (TEM), which revealedan average size of 13 nanometers, as shown in FIG. 6. Dynamic lightscattering was also used to confirm the size of the polymernanoparticles, as shown in FIG. 7.

To confirm the structure of the nanoparticles, FRET experiments wereperformed on the poly(oxanorborneneimide)undecyl trimethylammoniumanalog (Polymer 7 trimethylammonium analog (P7-TMA). By functionalizingP7-TMA polymers with dyes that exhibit FRET (a dye which acts as anelectron transfer agent (donor) and an electron acceptor agent(acceptor)) and upon mixing these two in aqueous environments, FRET wasobserved by the energy transfer from the donor to the acceptor. The FRETresults, as well as a schematic illustration of the FRET process, areshown in FIG. 8.

Biological Evaluation

Determination of Antimicrobial Activities of Cationic Polymers:

Bacteria were cultured in LB medium at 37° C. and 275 rpm untilstationary phase. The cultures were then harvested by centrifugation andwashed with 0.85% sodium chloride solution three times. Concentrationsof resuspended bacterial solution were determined by optical densitymeasured at 600 nanometers. M9 medium was used to make dilutions ofbacterial solution to a concentration of 1×10⁶ cfu/mL. A volume of 50microliters of these solutions was added into a 96-well plate and mixedwith 50 microliters of polymer solutions in M9 medium, giving a finalbacterial concentration of 5×10⁵ cfu/mL. Polymer concentration wasvaried according to a standard protocol, ranging from 1024 to 4nanomolar (nM). A growth control group without polymers and a sterilecontrol group with only growth medium were carried out at the same time.Incubation of the polymers with bacteria was performed for 16 hours.Cultures were performed in triplicates, and at least two independentexperiments were repeated on different days. The minimal inhibitorconcentration (MIC) is defined as the lowest concentration of polymerthat inhibits visible growth as observed with the unaided eye.

Determination of Hemolysis of Cationic Polymers:

Citrate-stabilized human whole blood (pooled, mixed gender) waspurchased from Bioreclamation LLC, NY and processed as soon as received.10 milliliters of phosphate buffered saline (PBS) was added to the bloodand centrifuged at 5000 rpm for 5 minutes. The supernatant was carefullydiscarded and the red blood cells (RBCs) were dispersed in 10milliliters of PBS. This step was repeated at least five times. Thepurified RBCs were diluted in 10 milliliters of PBS and kept on iceduring the sample preparation. 0.1 milliliter of RBC solution was addedto 0.4 milliliters of polymer solution in PBS in a 1.5 millilitercentrifuge tube (Fisher) and mixed gently by pipetting. RBCs incubatedwith PBS and water was used as negative and positive controls,respectively. All polymer samples as well as controls were prepared intriplicate. The mixture was incubated at 37° C. for 30 minutes whileshaking at 150 rpm. After incubation period, the solution wascentrifuged at 4000 rpm for 5 minutes and 100 microliters of supernatantwas transferred to a 96-well plate. The absorbance value of thesupernatant was measured at 570 nm using a microplate reader (SpectraMaxM2, Molecular devices) with absorbance at 655 nm as a reference. Thepercent hemolysis was calculated using the following formula:

${\% \mspace{14mu} {Hemolysis}} = {\frac{\left( {{{Sample}\mspace{14mu} {Absorbance}} - {{Negative}\mspace{14mu} {Control}\mspace{14mu} {Absorbance}}} \right)\mspace{14mu}}{\begin{pmatrix}{{{Positive}\mspace{14mu} {Control}\mspace{14mu} {Absorbance}} -} \\{{Negative}\mspace{14mu} {Control}\mspace{14mu} {Absorbance}}\end{pmatrix}}*100}$

Propidium Iodide Staining Assay:

E. coli CD-2, P. Aeruginosa ATCC19660 and MRSA CD-489 (1×10⁸ cfu/mL)were incubated with 1 μM Polymer 7 in M9 media at 37° C. and 275 rpm for3 hours. The bacteria solutions were then mixed with PI (2 μM) andincubated for 30 minutes in the dark. Five microliters of each samplewas placed on a glass slide with a glass coverslip and observed with aconfocal laser scanning microscope, Zeiss 510 (Carl Zeiss, Jena,Germany) using a 543 nm excitation wavelength.

Resistance Development:

E. coli CD-2 was inoculated in M9 medium with 85 nM (⅔ of 128 nM, MIC)of Polymer 7 at 37° C. and 275 rpm for 16 hours. The culture was thenharvested and tested for MIC as describe above. E. coli CD-2 wascultured without polymer as well every time as a control for comparisonof MICs.

Biofilm Formation and Treatment:

Bacteria were inoculated in lysogeny broth (LB) medium at 37° C. untilstationary phase. The cultures were then harvested by centrifugation andwashed with 0.85% sodium chloride solution three times. Concentrationsof resuspended bacterial solution were determined by optical densitymeasured at 600 nanometers. Seeding solutions were then made in M9medium to reach an OD600 of 0.1. A 100 microliter amount of the seedingsolutions was added to each well of the 96 well microplate. The plateswere covered and incubated at room temperature under static conditionsfor 1 day. The stock solution of polymers was then diluted to thedesired level and incubated with the biofilms for 3 hours at 37° C.Biofilms were washed with phosphate buffered saline (PBS) three timesand viability was determined using an Alamar Blue assay. Minimal M9medium without bacteria was used as a negative control.

Table 1 shows a summary of various polymers prepared according to theabove procedures, and the results of the biological evaluation of thecorresponding polymer nanoparticles.

TABLE 1 Molecular Selectivity Weight MIC (ug/ml) HC₅₀ (HC₅₀/MIC) Ex.Polymer R Group (kg/mol) P. aeruginose (ug/ml) P. aeruginose 1 9 Methyl20 2.5 402 160 2 9 Ethyl 20 2.7 417 160 3 9 Butyl 22 1.4 446 330 4 9Decyl 22 2.0 25 10 5 7 Methyl 29 0.9 4700 5000 6 7 Ethyl 30 1.9 97005000 7 7 Butyl 32 4.6 ND ND 8 7 Hexyl 34 2.4 ND ND 9 7 Benzyl 34 4.8 NDND “ND” means not determined.

The polymeric nanoparticles described above possess low minimalinhibitor concentrations (MICs) in the range of 0.1 to 5.0 ug/mL,specifically 0.5 to 5.0 ug/mL, towards pathogenic planktonic bacteriawhile maintaining remarkable hemolytic activities (therapeutic index:hemolysis (50%)/MIC˜5,000 for Example 5).

Polymer particles of the polymer of Example 5 were further testedagainst multiple uropathogenic clinical isolates, listed in Table 2, todemonstrate the broad spectrum activity of the particles. Particles fromthe Example 5 polymer suppressed bacterial proliferation atconcentrations ranging from 64 to 128 nanomolar. These polymers showedsimilar antimicrobial activity against 5 clinical isolates of E. coliwith different susceptibility to clinical antibiotics (resistant to 1-17drugs), indicating their ability to evade common mechanisms of bacterialresistance. Additionally, engineered polymers were effective againstclinical isolates of Gram-negative P. aeruginosa and E. cloacae complex.Similarly, Gram-positive strains of S. aureus were susceptible topolymer particles from Example 5 including the highly virulent strain ofmethicillin-resistant S. aureus (MRSA). Table 2, below, shows theminimum inhibitory concentrations and therapeutic indices of the polymerparticles against various uropathogenic clinical isolate bacterialstrains. Therapeutic indices are calculated with respect to red bloodcells.

TABLE 2 Strain Species MIC (nM) TI (HC₅₀/MIC) CD-23 P. aeruginosa 642300 CD-1006 P. aeruginosa 128 1200 CD-489 S. aureus (MRSA) 64 2300 CD-2E. coli 128 1200 CD-3 E. coli 64 2300 CD-19 E. coli 64 2300 CD-549 E.coli 128 1200 CD-496 E. coli 128 1200 CD-866 E. cloacae 128 1200 CD-1412E. cloacae 128 1200 CD-1545 E. cloacae 128 1200

The polymer nanoparticles were also observed to be particularlyeffective in disrupting the bacterial cell membrane, which, withoutwishing to be bound by theory, is believed to be due to the highlycationic and hydrophobic nature of the nanoparticles. This wasdemonstrated using a propidium iodide (PI) staining assay. PI onlystains cells having compromised cell membranes, allowing them to bindwith nucleic acids and generate red fluorescence. Pathogenic E. coli(CD-2), S. aureus (CD-489), and non-pathogenic P. aeruginosa(ATCC 19660)were treated with 1 micromolar solution of nanoparticles of Example 5for 3 hours at 37° C. and subsequently stained with PI before imaging.Confocal microscopy was used to show that the polymer nanoparticlemechanism of action leads to bacterial membrane disruption in all threespecies, regardless of membrane composition or pathogenicity.

In addition, the present inventors have demonstrated effective killingof pathogenic biofilms, not yet observed from other polymer researchers.The biofilm eradication data for the polymer of Example 5 in Table 1(i.e., oxanorbornene polymer backbone with a C₁₁ spacer and a trimethylammonium head group) is shown in FIG. 9-12. The Example 5 polymer wasincubated with the biofilm at varying concentrations, and bacterialviability was determined for each, as described above. FIG. 9-12generally demonstrate that polymer nanoparticle concentrations whichcause an increase in viability is likely due to the phenomenon called“hormesis”, a biological response to low exposures to toxins and otherstressors. As the concentration of polymer nanoparticles increases(e.g., to greater than 500 nanomolar), viability was observed todecrease indicating the killing of the bacteria within the biofilms. Ingeneral, the present inventors have observed that complete eradicationof the biofilms can occur at concentrations of at least 2 μM.

FIG. 9 shows that the Example 5 polymer could effectively eradicate theP. aeruginosa ATC-19660 biofilm at a concentration of greater than 640nM. FIG. 10 shows that the Example 5 polymer could effectively eradicatethe S. aureus (MRSA) biofilm at a concentration of greater than 2 μM.FIG. 11 shows that the Example 5 polymer could effectively eradicate theP. aeruginosa CD-1006 biofilm at a concentration of greater than 2 μM.FIG. 12 shows that the Example 5 polymer could effectively eradicate theE. cloacae CD-1412 biofilm at a concentration of greater than 1 μM.

As shown in FIG. 13, the polymer of Example 5 was also demonstrated tobe non-toxic to macrophages, with greater than 80% viability observedfrom polymer concentrations of 20 nM to 2 μM. Furthermore, as shown inFIG. 14, the polymer nanoparticles were not observed to cause macrophagecells to release inflammatory cytokine TNF-alpha, suggesting the polymernanoparticles are immunocompatible. LPS is used as a positive control,which induces cytokine expression in macrophages.

The ability to eradicate biofilms on human tissue or organs is also ofsignificant importance. An in vitro co-culture model of mammalianfibroblast cells with biofilms grown over them was used. First,compatibility of the polymer particles was tested using the Example 5polymer with NIH 3T3 fibroblast cells at similar concentrations used toeradicate the pre-formed biofilms. No significant toxicity was observed.Next, P. aeruginosa bacteria was seeded on a confluent monolayer of NIH3T3 fibroblast cells overnight to generate biofilms prior to treatment.The co-cultures were treated with the Example 5 polymer nanoparticlesfor 3 hours, washed, and the viabilities of both bacteria andfibroblasts were determined. As shown in FIG. 15, a 4-6-fold logreduction in bacterial colonies was observed at concentrations rangingfrom 7.5 to 15 micromolar, while fibroblast viability was maintained.

Bacteria are capable of acquiring resistance quickly towards antibioticsand other antimicrobials, minimizing their therapeutic prospects inclinical settings. The present inventors subjected uropathogenic E. coli(CD-2) to multiple serial passages of sub-MIC (66% of MIC)concentrations of Example 5 polymer nanoparticles to investigate ifresistance towards the present polymer nanoparticles occurs. Theresulting bacterial population was defined as the first generation,harvested, and its MIC was evaluated. Subsequently, a second generationwas produced by exposing first generation with 66% MIC dosage ofpolymers. As shown in FIG. 16, it was observed that even at the 20^(th)serial passage (about 1300 bacterial generations) of CD-2, E. coli wasstill susceptible to 128 nanomolar of Example 5 polymer nanoparticles,as compared to the zero generation. Similar experiments were conductedon ciprofloxacin (quinolone) and ceftazidime (β-lactam), clinicallyrelevant antibiotics. Respectively, there was a 33,000 and 4,200-foldincrease in the MICs of antibiotics against CD-2 E. coli. Thissignificant result indicates the killing mechanism of the presentpolymer nanoparticles significantly undermines the onset of resistancedevelopment in bacteria. Notably, the polymeric nanoparticles remainun-resistant towards bacteria longer than previously reportedpolymer-based nanomaterials (˜600 generations—A. baumannn FADDI-AB156)and comparable to a recently discovered and novel antibiotic,teixobactin (˜1,300 generations—S. aureus ATCC 29213).

Polymer Nanoparticles for Theranostics

The present inventors have further developed a method for preparingbiofilm theranostic agents from the above described polymernanoparticles. “Theranostic” as used herein means the polymernanoparticles are capable of serving both as a diagnostic tool, as wellas a therapeutic agent. The present inventors have taken advantage ofthe highly acidic environment of a bacterial biofilm, and conjugated afluorescent dye via a pH sensitive linkage to the polymer used toprepare the polymer nanoparticles. In the conjugated form, thefluorescence of the dye is quenched. Upon incubating the particles withthe biofilms, acid-mediated hydrolysis of the dye linker restoresfluorescence. This aspect of the present disclosure provides a new toolfor imaging a biofilm. A schematic representation of this aspect isillustrated in FIG. 17.

Fluorescently tagged polymers were prepared according to the samegeneral procedure described above, except that a comonomer including thefluorescent dye (e.g., BODIPY) was included. The resulting polymerincluded the trimethylammonium-containing monomer having an elevencarbon spacer and the dye-containing comomoner in a ratio of 68:7. Asdescribed above, the polymer was self-assembled into polymernanoparticles in aqueous solution, where the fluorescence of the dye wasobserved to be quenched. Upon exposure to an acidic environment (e.g.,pH 5.0), the dye was released from the polymer nanoparticles and thefree BODIPY dye fluorescence was observed.

Biofilms of E. coli DH5a were used to test the ability of thefluorescently tagged polymer nanoparticles to penetrate and image thebiofilms. 10′ bacterial cells/milliliter (DS Red exp. E. coli) wereseeded (2 milliliters in M9 media with 1 mM IPTG) in a confocal dish andwere allowed to grow, old media was replaced every 24 hours. After 3days media was replaced by 1 μM of the polymeric nanoparticles wereadded to biofilms and were incubated for 1 hour. After 1 hour, biofilmswere washed with PBS three times. Confocal microscopy images wereobtained on a Zeiss LSM 510 Meta microscope by using a 63× objective.The settings of the confocal microscope were as follows: green channel:λ_(ex)=488 nm and λ_(em)=BP 505-530 nm; red channel: λ_(ex)=543 nm andλ_(em)=LP 650 nm. Emission filters: BP=band pass, LP=high pass.

Confocal images are shown in FIG. 18. FIG. 18, top row, shows thefluorescence recovery of the dye as the polymer nanoparticles penetratethe biofilm. The negative control (polymer without dye, middle row)shows no green fluorescence. A negative control of dye only shows nogreen fluorescence due to lack of the presence of the dye after washing(i.e., lack of the ability of the dye alone to penetrate the biofilm).FIG. 19 shows the normalized fluorescence intensities with respect tobiofilm depth. From FIG. 19, it can be seen that polymer nanoparticlesincluding BODIPY dyes (“Polymer(+)”) generate fluorescence whiledye-free polymer nanoparticles (“Polymer(−)”) show no fluorescence.

Co-Delivery of Polymer Nanoparticles and Antibiotics for TherapeuticSynergy

As will be demonstrated by the following examples, the present inventorshave further discovered that co-incubation of polymer nanoparticles andan antibiotic, colistin, with planktonic bacteria or biofilms produces asynergistic therapeutic response. Importantly, enhanced colistin uptakeinto biofilms with nanoparticles were present was observed, determinedby an increase in colistin fluorescence within the biofilm (colistin waslabeled with a fluorophore for imaging purposes). Without wishing to bebound by theory, it is believed that the enhanced uptake of theantibiotic into the biofilm can be attributed to the ability of thepolymer nanoparticles to both penetrate the biofilm matrix and disruptbacterial membranes, providing colistin with an avenue to enter biofilmsand bind the membrane target.

To assess possible synergy between antibiotics and polymericnanoparticles, two-dimensional checkerboard titrations were performedusing a micro-dilution method. In 96-well plates, 2-fold dilutions ofantibiotics against a range of 2-fold dilutions of nanoparticles wereused to determine the MIC of the combinations. The concentrations ofnanoparticles were varied from their MIC to 1/32^(th) of their MIC.Similarly the concentrations of antibiotics were varied from their MICto 1/32^(th) of their MIC. The checkerboard titrations were performed ina set of three independent experiments, repeated on different days.

Antibiotic-nanoparticle interaction was determined by calculating thefractional inhibitory concentration of antibiotics (FIC Ab) and NPs (FICNP) according to the following equations:

FIC_(Ab)=(MIC of antibiotic and NP combination)÷(MIC of antibioticalone)

FIC_(NP)=(MIC of antibiotic and NP combination)÷(MIC of NP alone)

FICI_(combination)=FIC_(Ab)+FIC_(NP)

FIC_(Ab) was plotted against FIC_(NP). A concave curve indicatessynergy, whereas a convex curve indicates antagonism. Synergy wasdefined as FICI values≤0.5, antagonism by FICI values>4.0, and additiveinteraction by FICI values between >0.5 and 4.0. The experiments wereconducted across four different planktonic bacteria strains (E. clocaecomplex “CD-1412”; Pseudomonas aeruginosa “CD-40”; E. coli “CD-549”; andAcinetobacter species “CD-575”). A plot of the results is shown in FIG.20. As shown in FIG. 20, the polymer nanoparticles are therapeuticallysynergistic with membrane binding antibiotics such as colistin. In thepresence of polymer nanoparticles, colistin's uptake in biofilms isenhanced nearly 3.5 times. These results suggest that bacteria canbecome more susceptible to certain antibiotics when polymernanoparticles are co-added.

The uptake of colistin into the bacterial biofilms was also analyzed.10⁸ bacterial cells/ml (DS Red exp E. coli) were seeded (2 ml in M9media, 1 mM IPTG) in a confocal dish and were allowed to grow, and oldmedia was replaced every 24 hours. Testing solutions of coumarin-taggedpolymer (1-1.5 μM), rhodamine conjugated colistin (10-20 mg/L) and acombination of both were prepared. After 3 days media was replaced bythe prepared testing solutions and biofilms were incubated for 1 hour,biofilm samples incubated with only M9 media were used as control. After1 hour, biofilms were washed with PBS three times and confocalmicroscopy images were obtained on a Zeiss LSM 510 Meta microscope byusing a 63× objective. The results show that when polymer nanoparticlesare added along with colistin to biofilms, an enhancement in colistinuptake occurs (observed as green fluorescence arising from a rhodamine Glabel that was conjugated to the colistin for imaging purposed),determined using confocal microscopy.

Co-localization was observed between bacteria (red fluorescence),colistin (green fluorescence), and polymer nanoparticles (bluefluorescence). The confocal images of coumarin labeled polymer,rhodamine labeled colistin, and red fluorescent bacteria as well astheir overlays, and the control when no polymer is present are providedin FIG. 21 (scale bars are 30 μm, biofilms are E. coli DH5a). FIG. 22shows a plot of the quantitative analysis of the images shown in FIG.21. Using observed fluorescence from the rhodamine-labeled colistin, itcan be seen that, in the presence of the polymer, colistin uptake in thebiofilm is enhanced, compared to when no polymer is added. Integratingand comparing the area under each of these curves shows that colistinuptake is enhanced by about 3.4 times when the polymer is present.

The polymers, polymer nanoparticles, and methods of the presentdisclosure include at least the following embodiments.

Embodiment 1

A polymer nanoparticle comprising, a polymer comprising repeating unitsof formula (I), wherein X is independently at each occurrence —O—, —S—,—CH₂—, —(CR⁴R⁵)—, or

wherein R⁴ and R⁵ are independently at each occurrence a C₁₋₆ alkylgroup and R⁶ and R⁷ are independently at each occurrence hydrogen or aC₁₋₆ alkyl group; L¹ is independently at each occurrence a divalentgroup that is (—CH₂—)_(z), wherein z is an integer from 3 to 18; and R¹is independently at each occurrence an ammonium group, a phosphoniumgroup, a zwitterionic group, a carboxylate group, a sulfonate group, analkylene oxide group, or a combination thereof.

Embodiment 2

The polymer nanoparticle of embodiment 1, wherein the nanoparticle has adiameter of 1 to 100 nanometers.

Embodiment 3

The polymer nanoparticle of embodiment 1 or 2, wherein X is —O—.

Embodiment 4

The polymer nanoparticle of embodiment 1 or 2, wherein X is —CH₂—.

Embodiment 5

The polymer nanoparticle of any one of embodiments 1 to 4, wherein z isan integer from 6 to 12.

Embodiment 6

The polymer nanoparticle of any one of embodiments 1 to 5, wherein R¹ isan ammonium-containing group of formula (II), wherein R² is a C₁₋₁₂alkyl group or a C₇₋₂₀ alkylaryl group; and Y is bromide, chloride,fluoride, iodide, hydroxide, phosphate, sulfonate, carbonate, acetate,hexafluorophosphate, tetrafluoroborate, mesylate, trifluoroacetate,p-toluenesulfonate, or a combination thereof.

Embodiment 7

The polymer nanoparticle of any one of embodiments 1 to 6, wherein X is—O—; z is an integer from 6 to 12; and R¹ is an ammonium-containinggroup of formula (II), wherein R² is a C₁₋₆ alkyl group or a benzylgroup and Y is bromide, hydroxide, or a combination thereof.

Embodiment 8

The polymer nanoparticle of any one of embodiments 1 to 6, wherein X is—CH₂—; z is an integer from 6 to 12; and R¹ is an ammonium-containinggroup of formula (II), wherein R² is a C₁₋₁₀ alkyl group, and Y isbromide, hydroxide, or a combination thereof.

Embodiment 9

The polymer nanoparticle of any one of embodiments 1 to 8, wherein thepolymer further comprises repeating units of formula (VIII), wherein Xis independently at each occurrence —O—, —S—, —CH₂—, —(CR⁴R⁵)—, or

wherein R⁴ and R⁵ are independently at each occurrence a C₁₋₆ alkylgroup and R⁶ and R⁷ are independently at each occurrence hydrogen or aC₁₋₆ alkyl group; and L³ is independently at each occurrence a C₃₋₂₀alkyl group.

Embodiment 10

The polymer nanoparticle of any one of embodiments 1 to 9, wherein thepolymer further comprises repeating units of formula (IX), wherein X isindependently at each occurrence —O—, —S—, —CH₂—, —(CR⁴R⁵)—, or

wherein R⁴ and R⁵ are independently at each occurrence a C₁₋₆ alkylgroup and R⁶ and R⁷ are independently at each occurrence hydrogen or aC₁₋₆ alkyl group; L¹ is independently at each occurrence a divalentgroup that is (—CH₂—)_(z), wherein z is an integer from 3 to 18; Z isindependently at each occurrence a divalent group that is (—CH₂—)_(z),wherein z is an integer from 3 to 18; y is 0 or 1; and R⁸ is afluorescent group.

Embodiment 11

The polymer nanoparticle of embodiment 10, wherein the repeating unitsof formula (IX) are present in a molar ratio of repeating units offormula (I):repeating units of formula (IX) of 15:1 to 5:1.

Embodiment 12

The polymer nanoparticle of any one of claims 1 to 11, wherein thepolymer has a weight average molecular weight of 5,000 to 100,000 gramsper mole.

Embodiment 13

A method of making the polymer nanoparticle of any one of embodiments 1to 12, the method comprising combining the polymer comprising repeatingunits of formula (I) and an aqueous solution.

Embodiment 14

A polymer comprising repeating units of formula (I), wherein X isindependently at each occurrence —O—, —S—, —CH₂—, —(CR⁴R⁵)—, or

wherein R⁴ and R⁵ are independently at each occurrence a C₁₋₆ alkylgroup and R⁶ and R⁷ are independently at each occurrence hydrogen or aC₁₋₆ alkyl group; L¹ is independently at each occurrence a divalentgroup that is (—CH₂—)_(z), wherein z is an integer from 3 to 18; and R¹is independently at each occurrence an ammonium group, a phosphoniumgroup, a zwitterionic group, a carboxylate group, a sulfonate group, analkylene oxide group, or a combination thereof.

Embodiment 15

The polymer of embodiment 14, wherein X is —O—.

Embodiment 16

The polymer of embodiment 14, wherein X is —CH₂—.

Embodiment 17

The polymer of any one of embodiments 14 to 16, wherein z is an integerfrom 6 to 12.

Embodiment 18

The polymer of any one of embodiments 14 to 17, wherein R¹ is anammonium-containing group of formula (II), wherein R² is a C₁₋₁₂ alkylgroup or a C₇₋₂₀ alkylaryl group; and Y is bromide, chloride, fluoride,iodide, hydroxide, phosphate, sulfonate, carbonate, acetate,hexafluorophosphate, tetrafluoroborate, mesylate, trifluoroacetate,p-toluenesulfonate, or a combination thereof.

Embodiment 19

The polymer of any one of embodiments 14 to 18, wherein X is —O—; z isan integer from 6 to 12; and R¹ is an ammonium-containing group offormula (II), wherein R² is a C₁₋₆ alkyl group or a benzyl group and Yis bromide, hydroxide, or a combination thereof.

Embodiment 20

The polymer of any one of embodiments 14 to 18, wherein X is —CH₂—; z isan integer from 6 to 12; and R¹ is an ammonium-containing group offormula (II), wherein R² is a C₁₋₁₀ alkyl group, and Y is bromide,hydroxide, or a combination thereof.

Embodiment 21

The polymer of any one of embodiments 14 to 20, wherein the polymer hasa weight average molecular weight of 5,000 to 100,000 grams per mole.

Embodiment 22

The polymer of any one of embodiments 14 to 21, further comprisingrepeating units of formula (VIII), wherein X is independently at eachoccurrence —O—, —S—, —CH₂—, —(CR⁴R⁵)—, or

wherein R⁴ and R⁵ are independently at each occurrence a C₁₋₆ alkylgroup and R⁶ and R⁷ are independently at each occurrence hydrogen or aC₁₋₆ alkyl group; and L³ is independently at each occurrence a C₃₋₂₀alkyl group.

Embodiment 23

The polymer of any one of embodiments 14 to 22, wherein the polymerfurther comprises repeating units of formula (IX), wherein X isindependently at each occurrence —O—, —S—, —CH₂—, —(CR⁴R⁵)—, or

wherein R⁴ and R⁵ are independently at each occurrence a C₁₋₆ alkylgroup and R⁶ and R⁷ are independently at each occurrence hydrogen or aC₁₋₆ alkyl group; L¹ is independently at each occurrence a divalentgroup that is (—CH₂—)_(z), wherein z is an integer from 3 to 18; Z is adivalent C₆₋₂₀ arylene group, a divalent C₁₋₂₀ alkylene oxide group, adivalent poly(C₁₋₆ alkylene oxide) group, or an amino acid containinggroup; y is 0 or 1; and R⁸ is a fluorescent group.

Embodiment 24

A method of treating a bacterial biofilm, the method comprisingcontacting an aqueous composition comprising a plurality of polymernanoparticles according to any one of claims 1 to 12 or the polymer ofany one of claims 14 to 23 with a bacterial biofilm.

Embodiment 25

The method of embodiment 24, wherein the aqueous composition comprises0.0001 to 1 weight percent of the polymer nanoparticles; and 99 to99.9999 weight percent of an aqueous solution.

Embodiment 26

The method of any of embodiments 24 to 25, wherein the method furthercomprises contacting an antibiotic with the bacterial biofilm,preferably wherein the contacting of the antibiotic with the bacterialbiofilm occurs simultaneously with the contacting of the aqueouscomposition and the bacterial biofilm.

Embodiment 27

The method of any one of embodiments 24 to 26, wherein the bacterialbiofilm comprises Escherichia coli, Pseudomonas bacteria, Staphylococcalbacteria, Enterobacter bacteria, Streptococcus bacteria, Haemophilusinfluenzae, Leptospira interrogans, Legionella bacteria, Micrococcusbacteria, Bacillus bacteria, Burkholderia bacteria, Amycolatopsisbacteria, Mycobacterium bacteria, Acinetobacter bacteria, Enterococcusbacteria, Klebsiella bacteria, Acinetobacter bacteria, or a combinationthereof.

Embodiment 28

A method for detecting a bacterial biofilm, the method comprisingcontacting an aqueous composition comprising a plurality of polymernanoparticles comprising a copolymer comprising repeating units offormula (I) and (IX), wherein X is independently at each occurrence —O—,—S—, —CH₂—, —(CR⁴R⁵)—, or

wherein R⁴ and R⁵ are independently at each occurrence a C₁₋₆ alkylgroup and R⁶ and R⁷ are independently at each occurrence hydrogen or aC₁₋₆ alkyl group; L¹ is independently at each occurrence a divalentgroup that is (—CH₂—)_(z), wherein z is an integer from 3 to 18; R¹ isindependently at each occurrence an ammonium group, a phosphonium group,a zwitterionic group, a carboxylate group, a sulfonate group, analkylene oxide group, or a combination thereof; Z is a divalent C₆₋₂₀arylene group, a divalent C₁₋₂₀ alkylene oxide group, a divalentpoly(C₁₋₆ alkylene oxide) group, or an amino acid containing group; y is0 or 1; and R⁸ is a fluorescent group; with a surface; and measuringfluorescence, wherein the presence of fluorescence is indicative of thepresence of a bacterial biofilm.

This written description uses examples to disclose the invention,including the best mode, and also to enable any person skilled in theart to make and use the invention. The patentable scope of the inventionis defined by the claims, and may include other examples that occur tothose skilled in the art. Such other examples are intended to be withinthe scope of the claims if they have structural elements that do notdiffer from the literal language of the claims, or if they includeequivalent structural elements with insubstantial differences from theliteral language of the claims.

All cited patents, patent applications, and other references areincorporated herein by reference in their entirety. However, if a termin the present application contradicts or conflicts with a term in theincorporated reference, the term from the present application takesprecedence over the conflicting term from the incorporated reference.

All ranges disclosed herein are inclusive of the endpoints, and theendpoints are independently combinable with each other. Each rangedisclosed herein constitutes a disclosure of any point or sub-rangelying within the disclosed range.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. Further, it should further be noted that the terms “first,”“second,” and the like herein do not denote any order, quantity, orimportance, but rather are used to distinguish one element from another.The modifier “about” used in connection with a quantity is inclusive ofthe stated value and has the meaning dictated by the context (e.g., itincludes the degree of error associated with measurement of theparticular quantity).

As used herein, the term “alkyl” means a branched or straight chain,saturated, monovalent hydrocarbon group, e.g., methyl, ethyl, i-propyl,and n-butyl. “Alkylene” means a straight or branched chain, saturated,divalent hydrocarbon group (e.g., methylene (—CH₂—) or propylene(—(CH₂)₃—)). “Aryl” means a monovalent, monocyclic or polycyclicaromatic group (e.g., phenyl or naphthyl). Unless otherwise indicated,each of the foregoing groups can be unsubstituted or substituted,provided that the substitution does not significantly adversely affectsynthesis, stability, or use of the compound. The term “substituted” asused herein means that at least one hydrogen on the designated atom orgroup is replaced with another group, provided that the designatedatom's normal valence is not exceeded. When the substituent is oxo(i.e., ═O), then two hydrogens on the atom are replaced. Combinations ofsubstituents and/or variables are permissible provided that thesubstitutions do not significantly adversely affect synthesis or use ofthe compound. Groups that can be present on a substituted positioninclude (—NO₂), cyano (—CN), hydroxy (—OH), halogen, thiol (—SH),thiocyano (—SCN), C₂₋₆ alkanoyl (e.g., acyl (H₃CC(═O)—); carboxamido;C₁₋₆ or C₁₋₃ alkyl, cycloalkyl, alkenyl, and alkynyl (including groupshaving at least one unsaturated linkages and from 2 to 8, or 2 to 6carbon atoms); C₁₋₆ or C₁₋₃ alkoxy; C₆₋₁₀ aryloxy such as phenoxy; C₁₋₆alkylthio; C₁₋₆ or C₁₋₃ alkylsulfinyl; C₁₋₆ or C₁₋₃ alkylsulfonyl;aminodi(C₁₋₆ or C₁₋₃)alkyl; C₆₋₁₂ aryl having at least one aromaticrings (e.g., phenyl, biphenyl, naphthyl, or the like, each ring eithersubstituted or unsubstituted aromatic); C₇₋₁₉ arylalkyl having 1 to 3separate or fused rings and from 6 to 18 ring carbon atoms; orarylalkoxy having 1 to 3 separate or fused rings and from 6 to 18 ringcarbon atoms.

1. A polymer nanoparticle comprising, a polymer comprising repeatingunits of formula (I)

wherein X is independently at each occurrence —O—, —S—, —CH₂—,—(CR⁴R⁵)—, or

 wherein R⁴ and R⁵ are independently at each occurrence a C₁₋₆ alkylgroup and R⁶ and R⁷ are independently at each occurrence hydrogen or aC₁₋₆ alkyl group; L¹ is independently at each occurrence a divalentgroup that is (—CH₂—)_(z), wherein z is an integer from 3 to 18; and R¹is independently at each occurrence an ammonium group, a phosphoniumgroup, a zwitterionic group, a carboxylate group, a sulfonate group, analkylene oxide group, or a combination thereof.
 2. The polymernanoparticle of claim 1, wherein the nanoparticle has a diameter of 1 to100 nanometers.
 3. The polymer nanoparticle of claim 1, wherein X is —O—or —CH₂—; z is an integer from 6 to 12; and R¹ is an ammonium-containinggroup of formula (II)

wherein R² is a C₁₋₁₂ alkyl group or a C₇₋₂₀ alkylaryl group; and Y isbromide, chloride, fluoride, iodide, hydroxide, phosphate, sulfonate,carbonate, acetate, hexafluorophosphate, tetrafluoroborate, mesylate,trifluoroacetate, p-toluenesulfonate, or a combination thereof.
 4. Thepolymer nanoparticle of claim 1, wherein X is —O—; z is an integer from6 to 12; and R¹ is an ammonium-containing group of formula (II), whereinR² is a C₁₋₆ alkyl group or a benzyl group and Y is bromide, hydroxide,or a combination thereof.
 5. The polymer nanoparticle of claim 1,wherein X is —CH₂—; z is an integer from 6 to 12; and R¹ is anammonium-containing group of formula (II), wherein R² is a C₁₋₁₀ alkylgroup, and Y is bromide, hydroxide, or a combination thereof.
 6. Thepolymer nanoparticle of claim 1, wherein the polymer further comprisesrepeating units of formula (VIII)

wherein X is independently at each occurrence —O—, —S—, —CH₂—,—(CR⁴R⁵)—, or

 wherein R⁴ and R⁵ are independently at each occurrence a C₁₋₆ alkylgroup and R⁶ and R⁷ are independently at each occurrence hydrogen or aC₁₋₆ alkyl group; and L³ is independently at each occurrence a C₃₋₂₀alkyl group.
 7. The polymer nanoparticle of claim 1, wherein the polymerfurther comprises repeating units of formula (IX)

wherein X is independently at each occurrence —O—, —S—, —CH₂—,—(CR⁴R⁵)—, or

 wherein R⁴ and R⁵ are independently at each occurrence a C₁₋₆ alkylgroup and R⁶ and R⁷ are independently at each occurrence hydrogen or aC₁₋₆ alkyl group; L¹ is independently at each occurrence a divalentgroup that is (—CH₂—)_(z), wherein z is an integer from 3 to 18; Z is adivalent C₆₋₂₀ arylene group, a divalent C₁₋₂₀ alkylene oxide group, adivalent poly(C₁₋₆ alkylene oxide) group, or an amino acid containinggroup; y is 0 or 1; and R⁸ is a fluorescent group.
 8. The polymernanoparticle of claim 7, wherein the repeating units of formula (IX) arepresent in a molar ratio of repeating units of formula (I):repeatingunits of formula (IX) of 15:1 to 5:1.
 9. A polymer comprising repeatingunits of formula (I)

wherein X is independently at each occurrence —O—, —S—, —CH₂—,—(CR⁴R⁵)—, or

 wherein R⁴ and R⁵ are independently at each occurrence a C₁₋₆ alkylgroup and R⁶ and R⁷ are independently at each occurrence hydrogen or aC₁₋₆ alkyl group; L¹ is independently at each occurrence a divalentgroup that is (—CH₂—)_(z), wherein z is an integer from 3 to 18; and R¹is independently at each occurrence an ammonium group, a phosphoniumgroup, a zwitterionic group, a carboxylate group, a sulfonate group, analkylene oxide group, or a combination thereof.
 10. The polymer of claim9, wherein X is —O— or —CH₂—; z is an integer from 6 to 12; and R¹ is anammonium-containing group of formula (II)

wherein R² is a C₁₋₁₂ alkyl group or a C₇₋₂₀ alkylaryl group; and Y isbromide, chloride, fluoride, iodide, hydroxide, phosphate, sulfonate,carbonate, acetate, hexafluorophosphate, tetrafluoroborate, mesylate,trifluoroacetate, p-toluenesulfonate, or a combination thereof.
 11. Thepolymer of claim 9, wherein X is —O—; z is an integer from 6 to 12; andR¹ is an ammonium-containing group of formula (II), wherein R² is a C₁₋₆alkyl group or a benzyl group and Y is bromide, hydroxide, or acombination thereof.
 12. The polymer of claim 9, wherein X is —CH₂—; zis an integer from 6 to 12; and R¹ is an ammonium-containing group offormula (II), wherein R² is a C₁₋₁₀ alkyl group, and Y is bromide,hydroxide, or a combination thereof.
 13. The polymer of claim 9, whereinthe polymer has a weight average molecular weight of 5,000 to 100,000grams per mole.
 14. The polymer of claim 9, further comprising repeatingunits of formula (VIII)

wherein X is independently at each occurrence —O—, —S—, —CH₂—,—(CR⁴R⁵)—, or

 wherein R⁴ and R⁵ are independently at each occurrence a C₁₋₆ alkylgroup and R⁶ and R⁷ are independently at each occurrence hydrogen or aC₁₋₆ alkyl group; and L³ is independently at each occurrence a C₃₋₂₀alkyl group.
 15. The polymer of claim 9, wherein the polymer furthercomprises repeating units of formula (IX)

wherein X is independently at each occurrence —O—, —S—, —CH₂—,—(CR⁴R⁵)—, or

 wherein R⁴ and R⁵ are independently at each occurrence a C₁₋₆ alkylgroup and R⁶ and R⁷ are independently at each occurrence hydrogen or aC₁₋₆ alkyl group; L¹ is independently at each occurrence a divalentgroup that is (—CH₂—)_(z), wherein z is an integer from 3 to 18; Z is adivalent C₆₋₂₀ arylene group, a divalent C₁₋₂₀ alkylene oxide group, adivalent poly(C₁₋₆ alkylene oxide) group, or an amino acid containinggroup; y is 0 or 1; and R⁸ is a fluorescent group.
 16. A method oftreating a bacterial biofilm, the method comprising contacting anaqueous composition comprising a plurality of polymer nanoparticlesaccording to claim 1 with a bacterial biofilm.
 17. The method of claim16, wherein the aqueous composition comprises 0.0001 to 1 weight percentof the polymer nanoparticles; and 99 to 99.9999 weight percent of anaqueous solution.
 18. The method of claim 16, wherein the method furthercomprises contacting an antibiotic with the bacterial biofilm.
 19. Themethod of claim 16, wherein the bacterial biofilm comprises Escherichiacoli, Pseudomonas bacteria, Staphylococcal bacteria, Enterobacterbacteria, Streptococcus bacteria, Haemophilus influenzae, Leptospirainterrogans, Legionella bacteria, Micrococcus bacteria, Bacillusbacteria, Burkholderia bacteria, Amycolatopsis bacteria, Mycobacteriumbacteria, Acinetobacter bacteria, Enterococcus bacteria, Klebsiellabacteria, Acinetobacter bacteria, or a combination thereof.
 20. A methodfor detecting a bacterial biofilm, the method comprising: contacting anaqueous composition comprising a plurality of polymer nanoparticlescomprising a copolymer comprising repeating units of formula (I) and(IX)

wherein X is independently at each occurrence —O—, —S—, —CH₂—,—(CR⁴R⁵)—, or

 wherein R⁴ and R⁵ are independently at each occurrence a C₁₋₆ alkylgroup and R⁶ and R⁷ are independently at each occurrence hydrogen or aC₁₋₆ alkyl group; L¹ is independently at each occurrence a divalentgroup that is (—CH₂—)_(z), wherein z is an integer from 3 to 18; R¹ isindependently at each occurrence an ammonium group, a phosphonium group,a zwitterionic group, a carboxylate group, a sulfonate group, analkylene oxide group, or a combination thereof; Z is a divalent C₆₋₂₀arylene group, a divalent C₁₋₂₀ alkylene oxide group, a divalentpoly(C₁₋₆ alkylene oxide) group, or an amino acid containing group; y is0 or 1; and R⁸ is a fluorescent group; with a surface; and measuringfluorescence, wherein the presence of fluorescence is indicative of thepresence of a bacterial biofilm.